Arthritis Treatment

ABSTRACT

Administration of a monoclonal Ab (mAb) that specifically targets IL-1α is useful to treating articular and extra-articular symptoms of arthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the priority of U.S. provisional patentapplication Ser. No. 61/356,176 filed on Jun. 18, 2010, which isincorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention relates generally to the fields of immunology,inflammation, arthritis, and medicine. More particularly, the inventionrelates to the use of antibodies (Abs) which specifically bindinterleukin-1α (IL-1α) to treat one or more symptoms of arthritis.

BACKGROUND

Arthritis, the most common cause of disability in the United States, isa collection of different conditions such as osteoarthritis, rheumatoidarthritis, gout, psoriatic arthritis, septic arthritis, and reactivearthritis. All types of arthritis are characterized by jointinflammation which causes pain, swelling, redness, stiffness, and warmthat that affected site. Because afflicted subjects are less mobile due topain and stiffness, arthritis can indirectly lead to obesity, highcholesterol, and/or heart disease. Arthritis can also causeextra-articular disease such as iritis, uveitis, oral ulcers,inflammation of the gastrointestinal tract, inflammation of thegenitourinary tract, and skin lesions.

For most types of arthritis, no cure exists and treatment is largelysymptomatic, e.g., administration of analgesics and anti-inflammatorydrugs. Non-steroidal anti-inflammatory drugs (NSAIDs) can be used toreduce inflammation and pain. While generally effective, NSAIDs maycause side effects such as abdominal pain, bleeding, ulcers, and liverand kidney damage. Corticosteroids are effective at reducinginflammation and joint damage, but can cause a number of side effect arealso associated including bruising, weight gain, cataracts, bonethinning, diabetes, and hypertension. Other drugs commonly used to treatarthritis are methotrexate, cyclosporine, cyclophosphamide, leflunomide,hydroxychloroquine, sulfasalazine, and minocycline. These too can causeside effects such as liver damage and immunosuppression. Tumor necrosisfactor (TNF) inhibitors like etanercept (Enbrel), infliximab (Remicade),and adalimumab (Humira) are also useful for treating arthritis. Sideeffects of TNF inhibitors include injection site reactions, heartfailure, lymphoma, and increased risk of infection.

SUMMARY

The invention is based on the discovery that administration of anantibody (Ab) that specifically targets IL-1α in a human subjectsuffering from arthritis reduces the number of CD14+IL-1α+ peripheralblood monocytes in the subject and markedly ameliorates inflammation inboth articular and extraarticular sites—all without any observed sideeffects other than pain at the administration site.

Accordingly, the invention features a method of treating an inflammatorypathology associated with arthritis in a human subject by administeringto the subject a pharmaceutical composition including a pharmaceuticallyacceptable carrier and an amount of an anti-IL-1α antibody effective toreduce at least one symptom of the inflammatory pathology in thesubject. The symptom can be joint inflammation such as of the wrist orshoulder, or inflammation of the eye such as uveitis. The anti-IL-1αantibody can be a monoclonal antibody such as an IgG1. The anti-IL-1αantibody can be the monoclonal antibody designated as MABp1 or amonoclonal antibody that includes one or more complementaritydetermining regions (CDRs) of MABp1.

The pharmaceutical composition can be administered to the subject byinjection, subcutaneously, intravenously, intramuscularly,intraocularly, or directly into an inflamed joint. The antibody mightalso be administered to the eye topically. In the method, the amount ofthe anti-IL-1α antibody effective to reduce at least one symptom of theinflammatory pathology in the subject can be sufficient to raise thesubject's peripheral blood concentration of anti-IL-1α antibody to atleast 4 ug/ml; and/or sufficient to decrease the number of the subject'sCD14+IL-1α+ peripheral blood monocytes by at least 5%.

The method might also include a step of measuring the number ofCD14+IL-1α+ monocytes in the subject's peripheral blood afteradministration of the pharmaceutical composition, e.g., wherein the stepof measuring the number of CD14+IL-1α+ monocytes in the subject'speripheral blood is performed at least two different time points afteradministration of the pharmaceutical composition.

In another aspect, the invention features a method inducing monocytevacuolization in a subject by administering to the subject apharmaceutical composition including a pharmaceutically acceptablecarrier and an amount of an anti-IL-1α antibody effective to inducevacuole formation in monocytes.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Commonly understood definitions ofbiological terms can be found in Rieger et al., Glossary of Genetics:Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991;and Lewin, Genes V, Oxford University Press: New York, 1994. Commonlyunderstood definitions of medical terms can be found in Stedman'sMedical Dictionary, 27^(th) Edition, Lippincott, Williams & Wilkins,2000.

As used herein, an “antibody” or “Ab” is an immunoglobulin (Ig), asolution of identical or heterogeneous Igs, or a mixture of Igs. An“antibody” can also refer to fragments and engineered versions of Igssuch as Fab, Fab′, and F(ab′)₂ fragments; and scFv's, heteroconjugateAbs, and similar artificial molecules that employ Ig-derived CDRs toimpart antigen specificity. A “monoclonal antibody” or “mAb” is an Abexpressed by one clonal B cell line or a population of Ab molecules thatcontains only one species of an antigen binding site capable ofimmunoreacting with a particular epitope of a particular antigen. A“polyclonal antibody” or “polyclonal Ab” is a mixture of heterogeneousAbs. Typically, a polyclonal Ab will include myriad different Abmolecules which bind a particular antigen with at least some of thedifferent Abs immunoreacting with a different epitope of the antigen. Asused herein, a polyclonal Ab can be a mixture of two or more mAbs.

An “antigen-binding portion” of an Ab is contained within the variableregion of the Fab portion of an Ab and is the portion of the Ab thatconfers antigen specificity to the Ab (i.e., typically thethree-dimensional pocket formed by the CDRs of the heavy and lightchains of the Ab). A “Fab portion” or “Fab region” is the proteolyticfragment of a papain-digested Ig that contains the antigen-bindingportion of that Ig. A “non-Fab portion” is that portion of an Ab notwithin the Fab portion, e.g., an “Fc portion” or “Fc region.” A“constant region” of an Ab is that portion of the Ab outside of thevariable region. Generally encompassed within the constant region is the“effector portion” of an Ab, which is the portion of an Ab that isresponsible for binding other immune system components that facilitatethe immune response. Thus, for example, the site on an Ab that bindscomplement components or Fc receptors (not via its antigen-bindingportion) is an effector portion of that Ab.

When referring to a protein molecule such as an Ab, “purified” meansseparated from components that naturally accompany such molecules.Typically, an Ab or protein is purified when it is at least about 10%(e.g., 9%, 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,99.9%, and 100%), by weight, free from the non-Ab proteins or othernaturally-occurring organic molecules with which it is naturallyassociated. Purity can be measured by any appropriate method, e.g.,column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis. A chemically-synthesized protein or other recombinant proteinproduced in a cell type other than the cell type in which it naturallyoccurs is “purified.”

By “bind”, “binds”, or “reacts with” is meant that one moleculerecognizes and adheres to a particular second molecule in a sample, butdoes not substantially recognize or adhere to other molecules in thesample. Generally, an Ab that “specifically binds” another molecule hasa K_(d) greater than about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹²liters/mole for that other molecule.

A “therapeutically effective amount” is an amount which is capable ofproducing a medically desirable effect in a treated animal or human(e.g., amelioration or prevention of a disease or symptom of a disease).

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publicationsmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions willcontrol. In addition, the particular embodiments discussed below areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph and table showing the pharmacokinetics of MABp1 afteradministration to a human subject with reactive arthritis.

FIG. 2 is a series of graphs and histograms showing flow cytometricblood analyses after administration of MABp1 to a human subject withreactive arthritis.

FIG. 3 is a series of graphs showing flow cytometric blood analysesafter administration of MABp1 to a human subject with reactivearthritis.

DETAILED DESCRIPTION

The invention encompasses compositions and methods for treating asymptom or pathologic process associated with arthritis in a subject.The below described preferred embodiments illustrate adaptation of thesecompositions and methods. Nonetheless, from the description of theseembodiments, other aspects of the invention can be made and/or practicedbased on the description provided below.

General Methodology

Methods involving conventional immunological and molecular biologicaltechniques are described herein. Immunological methods (for example,assays for detection and localization of antigen-Ab complexes,immunoprecipitation, immunoblotting, and the like) are generally knownin the art and described in methodology treatises such as CurrentProtocols in Immunology, Coligan et al., ed., John Wiley & Sons, NewYork. Techniques of molecular biology are described in detail intreatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology,Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York.Ab methods are described in Handbook of Therapeutic Abs, Dubel, S., ed.,Wiley-VCH, 2007. General methods of medical treatment are described inMcPhee and Papadakis, Current Medical Diagnosis and Treatment 2010,49^(th) Edition, McGraw-Hill Medical, 2010; and Fauci et al., Harrison'sPrinciples of Internal Medicine, 17^(th) Edition, McGraw-HillProfessional, 2008

Treatment of Arthritis Symptoms

The compositions and methods described herein are useful for treating aninflammatory pathology associated with arthritis in a mammalian subjectby administering to the subject a pharmaceutical composition includingan amount of an anti-IL-1α antibody effective to reduce at least onesymptom of the inflammatory pathology in the subject. The mammaliansubject might be any that suffers from arthritis including, humanbeings, dogs, cats, horses, cattle, sheep, goats, and pigs. Humansubjects might be male, female, adults, children, seniors (65 andolder), and those with other diseases. The particular symptom orpathologic process associated with arthritis can be inflammation, pain,stiffness, or degeneration of a joint (e.g., in the wrist, digits[metacarpal or metatarsal joints], elbows, shoulders, hips, knees,ankles, foot, neck, or back) or extraarticular tissue (e.g., iritis,uveitis, oral ulcers, inflammation of the gastrointestinal tract,inflammation of the genitourinary tract, or skin lesions).

Antibodies and other Agents that Target IL-1α

Any suitable type of Ab that specifically binds IL-1α and reduces asymptom or pathologic process caused by arthritis in a subject might beused in the invention. For example, the anti-IL-1α Ab used might be mAb,a polyclonal Ab, a mixture of mAbs, or an Ab fragment or engineeredAb-like molecule such as an scFv. The Ka of the Ab is preferably atleast 1×10⁹ M⁻¹ or greater (e.g., greater than 9×10¹⁰ M⁻¹, 8×10¹⁰ M⁻¹,7×10¹⁰ M⁻¹, 6×10¹⁰ M⁻¹, 5×10¹⁰ M⁻¹, 4×10¹⁰ M⁻¹, 3×10¹⁰ M⁻¹, 2×10¹⁰ M⁻¹,or 1×10¹⁰ M⁻¹). In a preferred embodiment, the invention utilizes afully human mAb that includes (i) an antigen-binding variable regionthat exhibits very high binding affinity for human IL-1α and (ii) aconstant region that is effective at both activating the complementsystem though C1q binding and binding to several different Fc receptors.The human Ab is preferably an IgG1, although it might be of a differentisotype such as IgM, IgA, or IgE, or subclass such as IgG2, IgG3, orIgG4. One example of a particularly useful mAb is MABp1, anIL-1α-specific IgG1 monoclonal antibody described in U.S. patentapplication Ser. No. 12/455,458 filed on Jun. 1, 2009. Other useful mAbsare those than include at least one but preferably all the CDRs ofMABp1.

Because B lymphocytes which express Ig specific for human IL-1α occurnaturally in human beings, a presently preferred method for raising mAbsis to first isolate such a B lymphocyte from a subject and thenimmortalize it so that it can be continuously replicated in culture.Subjects lacking large numbers of naturally occurring B lymphocyteswhich express Ig specific for human IL-1α may be immunized with one ormore human IL-1α antigens to increase the number of such B lymphocytes.Human mAbs are prepared by immortalizing a human Ab secreting cell(e.g., a human plasma cell). See, e.g., U.S. Pat. No. 4,634,664.

In an exemplary method, one or more (e.g., 5, 10, 25, 50, 100, 1000, ormore) human subjects are screened for the presence of such humanIL-1α-specific Ab in their blood. Those subjects that express thedesired Ab can then be used as B lymphocyte donors. In one possiblemethod, peripheral blood is obtained from a human donor that possesses Blymphocytes that express human IL-1α-specific Ab. Such B lymphocytes arethen isolated from the blood sample, e.g., by cells sorting (e.g.,fluorescence activated cell sorting, “FACS”; or magnetic bead cellsorting) to select B lymphocytes expressing human IL-1α-specific Ig.These cells can then be immortalized by viral transformation (e.g.,using EBV) or by fusion to another immortalized cell such as a humanmyeloma according to known techniques. The B lymphocytes within thispopulation that express Ig specific for human IL-1α can then be isolatedby limiting dilution methods (e.g., cells in wells of a microtiter platethat are positive for Ig specific for human IL-1α are selected andsubcultured, and the process repeated until a desired clonal line can beisolated). See, e.g., Goding, Monoclonal Abs: Principles and Practice,pp. 59-103, Academic Press, 1986. Those clonal cell lines that expressIg having at least nanomolar or picomolar binding affinities for humanIL-1α are preferred. MAbs secreted by these clonal cell lines can bepurified from the culture medium or a bodily fluid (e.g., ascites) byconventional Ig purification procedures such as salt cuts, sizeexclusion, ion exchange separation, and affinity chromatography.

Although immortalized B lymphocytes might be used in in vitro culturesto directly produce mAbs, in certain cases it might be desirable to useheterologous expression systems to produce mAbs. See, e.g., the methodsdescribed in U.S. patent application Ser. No. 11/754,899. For example,the genes encoding an mAb specific for human IL-1α might be cloned andintroduced into an expression vector (e.g., a plasmid-based expressionvector) for expression in a heterologous host cell (e.g., CHO cells, COScells, myeloma cells, and E. coli cells). Because Igs include heavy (H)and light (L) chains in an H₂L₂ configuration, the genes encoding eachmay be separately isolated and expressed in different vectors.

Although generally less preferred due to the greater likelihood that asubject will develop an anti-Ab response, chimeric mAbs (e.g.,“humanized” mAbs), which are antigen-binding molecules having differentportions derived from different animal species (e.g., variable region ofa mouse Ig fused to the constant region of a human Ig), might be used inthe invention. Such chimeric Abs can be prepared by methods known in theart. See, e.g., Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851,1984; Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature,314:452, 1984. Similarly, Abs can be humanized by methods known in theart. For example, monoclonal Abs with a desired binding specificity canbe humanized by various vendors or as described in U.S. Pat. Nos.5,693,762; 5,530,101; or 5,585,089.

The mAbs described herein might be affinity matured to enhance orotherwise alter their binding specificity by known methods such as VHand VL domain shuffling (Marks et al. Bio/Technology 10:779-783, 1992),random mutagenesis of the hypervariable regions (HVRs) and/or frameworkresidues (Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813, 1994;Schier et al. Gene 169:147-155, 1995; Yelton et al. J. Immunol.155:1994-2004, 1995; Jackson et al., J. Immunol. 154(7):3310-9, 1995;and Hawkins et al, J. Mol. Biol. 226:889-896, 1992. Amino acid sequencevariants of an Ab may be prepared by introducing appropriate changesinto the nucleotide sequence encoding the Ab. In addition, modificationsto nucleic acid sequences encoding mAbs might be altered (e.g., withoutchanging the amino acid sequence of the mAb) for enhancing production ofthe mAb in certain expression systems (e.g., intron elimination and/orcodon optimization for a given expression system). The mAbs describedherein can also be modified by conjugation to another protein (e.g.,another mAb) or non-protein molecule. For example, a mAb might beconjugated to a water soluble polymer such as polyethylene glycol or acarbon nanotube (See, e.g., Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005). See, U.S. patent application Ser. No. 11/754,899.

Preferably, to ensure that high titers of human IL-1α-specific mAb canbe administered to a subject with minimal adverse effects, the mAbcompositions of the invention are at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,95, 96, 97, 98, 99, 99.9 or more percent by weight pure (excluding anyexcipients). The mAb compositions of the invention might include only asingle type of mAb (i.e., one produced from a single clonal B lymphocyteline) or might include a mixture of two or more (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10 or more) different types of mAbs.

To modify or enhance their function, the human IL-1α mAbs might beconjugated another molecule such as a cytotoxin. A human IL-1α specificmAb might be conjugated with one or more cytotoxins to more effectivelykill cells expressing IL-1α. Cytotoxins for use in the invention can beany cytotoxic agent (e.g., molecule that can kill a cell aftercontacting the cell) that can be conjugated to a human IL-1α specificmAb. Examples of cytotoxins include, without limitation, radionuclides(e.g., ³⁵S, ¹⁴C, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁸⁹Zr, ²⁰¹Tl, ¹⁸⁶Re, ¹⁸⁸Re, ⁵⁷Cu,²¹³Bi, and ²¹¹At), conjugated radionuclides, and chemotherapeuticagents. Further examples of cytotoxins include, but are not limited to,antimetabolites (e.g., 5-fluorouricil (5-FU), methotrexate (MTX),fludarabine, etc.), anti-microtubule agents (e.g., vincristine,vinblastine, colchicine, taxanes (such as paclitaxel and docetaxel),etc.), alkylating agents (e.g., cyclophasphamide, melphalan,bischloroethylnitrosurea (BCNU), etc.), platinum agents (e.g., cisplatin(also termed cDDP), carboplatin, oxaliplatin, JM-216, CI-973, etc.),anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibioticagents (e.g., mitomycin-C), topoisomerase inhibitors (e.g., etoposide,tenoposide, and camptothecins), or other cytotoxic agents such as ricin,diptheria toxin (DT), Pseudomonas exotoxin (PE) A, PE40, abrin, saporin,pokeweed viral protein, ethidium bromide, glucocorticoid, anthrax toxinand others. See, e.g., U.S. Pat. No. 5,932,188.

While the IL-1α specific Abs described above are preferred for use theinvention, in some cases, other agents that specifically target IL-1αmight be used so long as their administration leads to improvement ofone or more symptoms of arthritis. These other agents might includesmall organic molecules, aptamers, peptides, and proteins thatspecifically bind IL-1α.

Pharmaceutical Compositions and Methods

The anti-IL-1α Ab compositions may be administered to animals or humansin pharmaceutically acceptable carriers (e.g., sterile saline), that areselected on the basis of mode and route of administration and standardpharmaceutical practice. A list of pharmaceutically acceptable carriers,as well as pharmaceutical formulations, can be found in Remington'sPharmaceutical Sciences, a standard text in this field, and in USP/NF.Other substances may be added to the compositions and other steps takento stabilize and/or preserve the compositions, and/or to facilitatetheir administration to a subject.

For example, the Ab compositions might be lyophilized (see Draber etal., J. Immunol. Methods. 181:37, 1995; and PCT/US90/01383); dissolvedin a solution including sodium and chloride ions; dissolved in asolution including one or more stabilizing agents such as albumin,glucose, maltose, sucrose, sorbitol, polyethylene glycol, and glycine;filtered (e.g., using a 0.45 and/or 0.2 micron filter); contacted withbeta-propiolactone; and/or dissolved in a solution including amicrobicide (e.g., a detergent, an organic solvent, and a mixture of adetergent and organic solvent.

The Ab compositions may be administered to animals or humans by anysuitable technique. Typically, such administration will be parenteral(e.g., intravenous, subcutaneous, intramuscular, or intraperitonealintroduction). The compositions may also be administered directly to thetarget site (e.g., an inflamed joint, or the uvea or conjuctiva) by, forexample, injection or topical application. Other methods of delivery,e.g., liposomal delivery or diffusion from a device impregnated with thecomposition, are known in the art. The composition may be administeredin a single bolus, multiple injections, or by continuous infusion (e.g.,intravenously or by peritoneal dialysis).

A therapeutically effective amount is an amount which is capable ofproducing a medically desirable result in a treated animal or human. Aneffective amount of anti-IL-1α Ab compositions is an amount which showsclinical efficacy in arthritis patients as measured by the improvementin pain and function as well as the prevention of structural damage. Asis well known in the medical arts, dosage for any one animal or humandepends on many factors, including the subject's size, body surfacearea, age, the particular composition to be administered, sex, time androute of administration, general health, and other drugs beingadministered concurrently. A preferred dose is one that is sufficient toraise the subject's peripheral blood concentration of anti-IL-1α Ab toat least 4 (e.g., at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100,200, 300, 400, 500, 1000, 2500, or 5000) micrograms/ml. It is expectedthat an appropriate dosage of Abs would be in the range of about 0.2 to20 (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, or 100)mg/kg body weight for subcutaneous administration and about 0.001 to 50(e.g., 0.001, 0.01, 1, 5, 10, 15, 25, or 50) mg per eye for topicaladministration to the eye. The dose may be given repeatedly, e.g.,hourly, daily, weekly, or monthly.

EXAMPLES Example 1 Xilonix™

Xilonix™ is a sterile injectable liquid formulation of 15 mg/mL MABp1 ina stabilizing isotonic buffer (pH 6.4). Each 10-mL Type I borosilicateglass serum vial contains 5 mL of the formulation, and is sealed with a20-mm Dalkyo Fluorotec butyl rubber stopper and flip-off aluminum seal.The product is stored at 5±3° C., with excursions to room temperaturepermitted. The exact composition of the drug product is shown below:

Composition of the Drug Product(Xilonix ™) Ingredient Grade ManufacturerConcentration MABp1 antibody GMP XBiotech 15 mg/mL sodium phosphatedibasic compendial JT Baker 12 mg/mL citric acid monohydrate compendialJT Baker 2 mg/mL Trehalose.2H2O (high- compendial Ferro- 60 mg/mL puritylow endotoxin) Pfanstiehl polysorbate 80 compendial JT Baker 0.2 mg/mLPhosphoric acid, to adjust pH compendial JT Baker 0.04 mg/mL water forinjection compendial Microbix q.s.

Example 2 Treatment of Reactive Arthritis with an IL-1α-SpecificMonoclonal Antibody

A 48 year-old male patient with reactive arthritis was administered atotal 220 milligrams of MABp1, an IL-1α-specific monoclonal antibodydescribed in U.S. patent application Ser. No. 12/455,458 filed on Jun.1, 2009. The patient had a long history of reactive arthritis, startingat age 16, when he was diagnosed with Reiter's syndrome duringhospitalization for severe inflammation in his left knee. Thisinflammation resolved, yet the patient experienced periodic relapses inseveral joints until his mid-twenties. No further episodes occurreduntil, at age 35, the patient had a severe unilateral episode of uveitisthat lasted for 8 weeks. The uveitis was poorly managed with ophthalmiccorticosteroids and oral NSAIDS, resulting in some scaring. The patientsubsequently experienced at least three additional episodes of uveitisof varying intensities, one episode requiring subcorneal injection ofcorticosteroids.

Just prior to his 48^(th) birthday, the patient developed severe pain inhis left shoulder and wrist. Evident swelling and redness with almostcomplete loss of mobility affected the wrist. The patient was unable toabduct his left arm greater than about 20° due to intense shoulder pain.On that day, the patient was given a subacromial injection ofcorticosteroids into the left shoulder. The patient reported that thecondition continued to worsen with pain from shoulder and wristreportedly becoming continuous, interrupting work and preventing sleep.In addition, pain and irritation in the left eye ensued, indicatingonset of an episode of uveitis. This was reportedly the first time jointinflammation and uveitis occurred together. The patient was takingophthalmic corticosteroids, oral and topical ophthalmic NSAIDS withlittle apparent benefit.

On day 0 (forty-two days after the subacromial injection ofcorticosteroids), the patient was administered four subcutaneousinjections of MABp1, delivering a total of 110 mg of MABp1 (in equaldoses). No side effects other than pain during injection was reported.Blood was drawn by venous puncture immediately prior to injection intotwo 5 ml sodium heparin tubes. Plasma analysis using an enzyme-linkedimmunadsorbant assay (ELISA) for the detection of existing endogenousanti-IL-1α antibodies revealed no pre-existing antibodies.

On day 1, the patient reported that he woke up that morning without thethrobbing pain that had become the “first sensation upon waking” Overthe next several days there was an evident improvement in mobility.There was no induration or redness at the injection sites. A blood drawwas taken and flow cytometric analysis (FACS) was performed to evaluateleuckocyte subsets and IL-1α expression on monocytes. Analysis was alsoperformed on plasma to determine levels of MABp1 and to begin collectionof pharmacokinetic (pK) data for MABp1. FACS analysis of PBMC revealedthat most CD14+ monocytes (72.6%) expressed IL-1α. A MABp1 plasmaconcentration of 3.2 μg/ml was observed.

On day 6, another blood sample was taken and analyzed using FACS and forMABp1. The frequency of CD14+ monocytes stained by MABp1 had declined to47.3%. Plasma levels of MABp1 had increased to 7 μg/ml. Although notconfirmed, the increase in MABp1 concentration was considered to reflecta depot effect of the subcutaneous administration of MABp1. Althoughthere had been improvement, the patient still exhibited considerabletenderness and pain with movement and the uveitis had flared since theprevious weekend, where the patient had attended a party and consumedalcohol. The patient was administered another 110 mg of MABp1subcutaneously.

On day 14, a blood sample was taken and analyzed using FACS and pKanalysis was performed on plasma. CD14+ monocyte frequency stained byMABp1 further declined to 21.7%. However, plasma levels of MABp1 hadalso declined to 5.8 μg/ml. This was unanticipated, since plasma levelsof MABp1 had increased over the week after the first injection.

Approximately one month after the first injection of MABp1 the patientwas reevaluated. Marked improvement was noted in mobility and there wasno pain in the wrist. Pain in shoulder was present only upon abductionto 90°. FACS analysis revealed no detectable CD14+ monocytes stained byMABp1. Plasma levels of MABp1 had declined to 1.6 μg/ml, suggesting ahalf-life for MABp1 of about two weeks.

Over the course of the next several weeks the patient showed gradual butcontinuous improvement in mobility. There was complete resolution of theuveitis. The improvement was noted even though the patient discontinueduse of all medications after the first injection of MABp1. Approximatelythree months after the first injection of MABp1, the frequency of CD14+monocytes stained by MABp1 had returned to pre-treatment levels. MABp1levels in plasma declined to 0.07 μg/ml. However, the patient continuedto do well with continuing improvement in mobility of the shoulder.

Example 3 Screening of Plasma Samples for Endogenous AutoantibodyAgainst hIL-1A and Pharmacokinetics of MABp1

A method was developed for the screening of plasma samples forendogenous autoantibody against human IL-1α (hIL-1α) using a directELISA. This method was also used to determine pharmacokinetics (pK) ofMABp1 after administration, with the exception, that higher dilutionsplasma samples were made.

The direct ELISA involves coating of recombinant human IL-1α on apolystyrene microplate. The bound human IL-1A captures endogenousanti-human IL-1α antibody from test samples. An HRP-conjugated-Fcspecific, mouse-anti-human IgG is then used to detect the capturedendogenous anti-human IL-1A antibody, followed by treatment with TMBsubstrate. On reacting with HRP enzyme, the TMB substrate produces adeep blue-colored soluble product. The enzymatic reaction is stopped bythe addition of a stop solution that turns the blue-colored product toyellow. The colorimetric measurements are carried out on a microplatereader at 450 nm.

About 5 ml plasma sample per sample is provided. Plasma is kept at 2-8°C. prior to aliquoting and storage at −80° C. Plasma samples are diluted1:500, 1:1000 and 1:2000-fold to use as samples. A positive control inbuffer is used containing 20 μg/ml MABp1 antibody stock as 1:5,000 and1:10,000-fold dilutions on microplate. Buffer is used as a negativecontrol as well as a pre-determined negative control plasma, which isdiluted as 1:1,000,1:2,000 and 1:5,000. An additional positive plasmacontrol is used, which is plasma spiked with 20 μg/ml MABp1 antibody anddiluted as 1:5,000 and 1:10,000 for samples on the microplate.

If the positive control value falls within ±2 standard deviation, theELISA data is considered acceptable. However if the QC positive controlvalue falls beyond ±2 standard deviation, the ELISA data is consideredunacceptable and the experiment would be repeated. Using a Kaleidagraph,the logarithmic mean absorbance of standard solution is plotted as afunction of logarithmic concentration along with absorbance error bars.The standard curve should exhibit a linear behavior. Results from apharmacokinetics analysis of samples taken from the patient as describedin Example 2 are shown in FIG. 1.

Example 4 Flow Cytometric (FACS) Examination of Blood Lineage Subsets

FACS procedures are described for both whole blood staining, andstaining of peripheral blood mononuclear cells (PBMC) enriched fromwhole blood. Both whole blood and PBMC staining was performed on allsamples. This FACS analysis allows relative percentage determination ofblood lineage subsets: B and T lymphocytes, NK cells, monocytes,neutrophils, and IL-1α+ cells. Results from FACs analyses of samplestaken from the patient as described in Example 2 are shown in FIGS. 2and 3. A photomicrograph of a blood smear showed that MABp1administration caused extensive vacuolization in peripheral bloodmonocytes when analyzed 32 days post administration.

Example 5 Treatment of Uveitis with an IL-1α-specific MonoclonalAntibody

About two months following resolution of the uveitis described inExample 2, the patient experienced another episode of uveitis(predominantly iritis). The patient was started on corticoseroid andnon-steroidal anti-inflammatory drops (NSAIDS). Oral NSAIDS were alsoused. The uveitis was unresponsive to treatment and progressed. However,there was no evidence of any joint involvement, with shoulder continuingto show improvement in mobility. The patient was administered MABp1topically to the affected eye. MABp1 (15 mg/ml solution) wasadministered at a rate of one drop per minute, for ten minutes, for atotal of ten drops to the affected eye (approximately 3.75 mg in 0.25ml). The patient did not complain of any pain during the administration.However, for several hours after, the patient reported discomfort andburning. Oral NSAIDs were taken and the patient slept. The next morning,the patient reported considerable improvement, reduced pain and lessinflammation than prior to administration. Twenty-four hours after thefirst administration of the MABP1 drops, the patient administered 10drops in the same fashion. Again, discomfort and burning was noted. OralNSAIDs were taken, and again the patient took bed rest. The uveitisresolved itself completely. No further medications were taken. Norecurrence of uveitis was observed over the next four months.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for treating an inflammatory pathology associated witharthritis in a human subject, the method comprising the step ofadministering to the subject a pharmaceutical composition comprising apharmaceutically acceptable carrier and an amount of an anti-IL-1αantibody effective to reduce at least one symptom of the inflammatorypathology in the subject.
 2. The method of claim 1, wherein the at leastone symptom is joint inflammation.
 3. The method of claim 2, wherein thejoint inflammation is in the wrist of the subject.
 4. The method ofclaim 2, wherein the joint inflammation is in the shoulder of thesubject.
 5. The method of claim 1, wherein the at least one symptom isinflammation of the eye.
 6. The method of claim 5, wherein theinflammation is uveitis.
 7. The method of claim 1, wherein theanti-IL-1α antibody is a monoclonal antibody.
 8. The method of claim 7,wherein the monoclonal antibody is an IgG1.
 9. The method of claim 7,wherein the monoclonal antibody comprises a complementarity determiningregion of MABp1.
 10. The method of claim 7, wherein the monoclonalantibody is MABp1.
 11. The method of claim 1, wherein the pharmaceuticalcomposition is administered by injection.
 12. The method of claim 11,wherein the pharmaceutical composition is injected into an inflamedjoint.
 13. The method of claim 5, wherein the pharmaceutical compositionis administered to the eye topically.
 14. The method of claim 5, whereinthe pharmaceutical composition is administered to the eye by injection.15. The method of claim 1, wherein the amount of the anti-IL-1α antibodyeffective to reduce at least one symptom of the inflammatory pathologyin the subject is sufficient to raise the subject's peripheral bloodconcentration of anti-IL-1α antibody to at least 4 micrograms/ml.
 16. Amethod of inducing monocyte vacuolization in a subject, the methodcomprising administering to the subject an amount of an anti-IL-1αantibody effective to induce monocyte vacuolization in the subject. 17.The method of claim 16, wherein the subject has arthritis.
 18. Themethod of claim 16, wherein the anti-IL-1α antibody is a monoclonalantibody.
 19. The method of claim 18, wherein the monoclonal antibody isan IgG1.
 20. The method of claim 18, wherein the monoclonal antibodycomprises a complementarity-determining region of MABp1.
 21. The methodof claim 20, wherein the monoclonal antibody is MABp1.
 22. The method ofclaim 16, wherein the amount of the anti-IL-1α antibody administered tothe subject is sufficient to raise the subject's peripheral bloodconcentration of anti-IL-1α antibody to at least 4 micrograms/ml.